The long term goal of this project is a molecular understanding of the mechanisms by which neurotransmitters modulate ion channels through second messenger cascades and thus regulate transmitter release from presynaptic terminals. Modulation of transmitter release is an important form of plasticity involved in learning and memory. Moreover, pathophysiological changes in modulatory transmitter actions are thought to underlie certain neurologic and psychiatric diseases. The specific goal of this study is to investigate the molecular bases for the antagonistic modulation by serotonin (5-HT) and the neuropeptide FMRFamide of the background conductance S-K channel in mechanoreceptor sensory neurons of Aplysia. In Aplysia, 5-HT causes presynaptic facilitation of transmitter release from sensory terminals whereas FMRFamide causes presynaptic inhibition. These transmitters also exert antagonistic actions at the single channel level: 5-HT closes S channels whereas FMRFamide increases S channel opening. The changes in S-K current are thought to alter Ca influx into the terminals and alter release indirectly. This action of 5-HT is mediated by cAMP-dependent protein kinase (cAMP-PK). The action of FMRFamide is mediated by the 12-lipoxygenase metabolite of arachidonic acid, 12-HPETE. FMRFamide also reopens S channels closed by 5-HT or cAMP and causes protein dephosphorylation. The specific goals of this project are to study the molecular mechanisms whereby 5-HT and FMRFamide modulate S channel function and address the following questions: Do the actions of cAMP and/or 12-HPETE depend on the down- or up-modulation of phosphatase activity? Is 12-HPETE the final active metabolite or are downstream metabolites required? Where in the membrane is the 12-HPETE receptor located? Might 12-HPETE act as a first messenger to alter the activity of neighboring cells? The above questions will be addressed using a combined biochemical and electrophysiological approach. Single S channel currents will be recorded in cell-free patches and whole cell S currents recorded under voltage clamp. Purified kinases, phosphatases, and various inhibitors will be applied to patches or injected into sensory neurons to determine how they alter S channel function or interact with the modulatory actions of cAMP-PK and arachidonate metabolites. Parallel biochemical assays of phosphatase activity and modulation of this activity in homogenates of sensory neuron clusters will also be performed.